Carotenoids are pigments which are found in all photo-synthetic organisms. They play an important role as components of the photosynthetic reaction center and in mediating protection against photooxidative damage.
The carotenoid biosynthetic pathway proceeds from geranylgeranyl diphosphate (GGDP) to astaxanthin. As the result of the condensation of two molecules of geranylgeranyl pyrophosphate, the phytoene synthase enzyme (PSY) forms the C40 structure phytoene. Starting from phytoene, the phytoene desaturase enzyme (PDS) synthetizes ζ-carotene by eliminating protons and incorporating two double bonds. The intermediate of this synthetic step is phytofluene. ζ-Carotene, in turn, is converted into lycopene in a two-step desaturation reaction, which proceeds via the intermediate neurosporin. The enzyme which is responsible therefor is ζ-carotene desaturase (ZDS). Lycopene is converted into β-carotene via a lycopene cyclase (LCYB). Starting from β-carotene, two further enzymes are involved in the formation of astaxanthin. Firstly, the β-carotene ketolase enzyme (BKT) introduces in each case one keto group at the 4 and the 4′ position. Secondly, in each case one hydroxyl group is attached at the 3 and 3′ positions on the ion ring of the astaxanthin precursor via the carotenoid hydroxylase enzyme (CHY).
Overexpressing a bacterial phytoene synthase from Eriwinia uredovora has made it possible to influence, or stimulate, carotenoid biosynthesis in transgenic tomato plants and thus to increase the amount of carotenoid synthesized by a factor of 2-4 (Fraser, Romer et al. 2002).
However, it is the enzyme phytoene desaturase which plays the central key role in the carotenoid biosynthetic pathway (FIG. 1). The pds genes, which code for phytoene desaturase, have been cloned from cyanobacteria (Chamovitz et al., 1991; Martínez-Férez and Vioque, 1992; Martínez-Férez et al.,. 1994) and higher plants (Bartley et al., 1991; Pecker et al., 1992) and are successfully overexpressed in E. coli. The bleaching herbicide norflurazon is known as a reversible, noncompetitive inhibitor of phytoene desaturase. Mutated forms of phytoene desaturase have been described for the cyanobacterium Synechococcus; they confer resistance to norflurazon to the bacterium. In each case, the resistance-imparting mutations are based on a single amino acid substitution. Within the scope of the mutation studies carried out in Synechococcus, resistances have been found for the following amino acid substitutions: Arg195Pro; Leu320Pro; Val403Gly; Leu437Arg (Linden et al., 1990; Chamovitz et al., 1993). Despite the fact that norflurazon and other such bleaching herbicides have been used for some time for controlling weeds, no resistant naturally occurring plants have been isolated to date.
Among the intermediates and products of the carotenoid biosynthetic pathway, it is in particular the keto carotenoid astaxanthin which is of commercial importance. Astaxanthin has a higher antioxidant activity than other intermediates of the carotenoid biosynthetic pathway. Astaxanthin acts as a quencher of free radicals and active oxygen species (Kobayashi and Sakamoto, 1999), as an enhancer of immune responses (Jyonouchi et al., 1995) and as an anticancer agent (Tanaka et al., 1994, 1995). Owing to its natural effect as potent antioxidant, astaxanthin is also employed as food supplement. It is used as food additive with colorant effect in fish farming.
Some green algae such as, for example, Dunaliella bardawil and Haematococcus pluvialis have the unique ability of accumulating carotenoids under stress conditions. In this context, it is in particular H. pluvialis which is suitable for the natural production of astaxanthin. H. pluvialis is capable of accumulating astaxanthin in amounts of up to 4% of its dry weight. This is why H. pluvialis plays a key role in the commercial production of astaxanthin, since the chemico-synthetic route for the production of astaxanthin is among the most complex which are commercially employed for producing an active ingredient.
The genetic transformation of green algae has been described repeatedly for Chlamydomonas reinhardtii and some Chlorella species (Kindle 1990; Lumbreras et al., 1998; Hawkins and Nakamura, 1999; Kim et al., 2002). Owing to their metabolism, these two algal species are thought to be less interesting for the natural production of carotenoids.
The genetic transformation of H. pluvialis has been described by Teng at al. (2003). The β-galactosidase reporter gene lacZ under the control of the SV40 promoter has been integrated into the algal genome by means of microparticle bombardment. Successfully transformed algal cells must be detected and selected individually with the aid of optical means. Algal cells which are transformed with the lacZ gene have no resistance to toxically, or dominantly, acting selection agents.
Although astaxanthin biosynthesis has been studied in detail during the last 10 years (Lu et al., 1995; Fraser et al., 1998), the production of astaxanthin by H. pluvialis on the large scale of the biotechnology industry remains problematic since, inter alia, cell division is inhibited during astaxanthin biosynthesis (Boussiba and von Vonshak, 1991).
It is an object of the present invention to provide a DNA vector which can be used for suitably genetically modifying the genome of eukaryotic cells, in particular of algae such as H. pluvialis, in order to be able to influence, or increase, the in-vivo synthesis of natural carotenoids and isoprenoids and, in particular, astaxanthin biosynthesis. It is another object of the invention to provide a vector which can be employed as dominantly-selective marker for the transformation in eukaryotic cells, in particular of algae such as H. pluvialis, and which makes it possible to select, in a simple manner, successfully transformed eukaryotic cells, in particular of algae such as H. pluvialis. 
The invention achieves this object by a vector comprising (a) a DNA sequence which codes for the protein phytoene desaturase which has a resistance-conferring amino acid substitution at one position, and (b) a multiple cloning site into which any DNA sequence can be cloned.
In this context, the vectors according to the invention comprise any DNA molecules which can be used as vehicles with the aid of which foreign DNA can be introduced into a cell. They encompass cosmids, phages, viruses, YACs, BACs, more linear DNA molecules and, in particular, circular plasmids.
Within the scope of the invention, the DNA sequence of the enzyme phytoene desaturase (PDS) from H. pluvialis has been isolated from a genomic DNA library and sequenced (SEQ ID NO:1). The corresponding protein sequence of the H. pluvialis phytoene desaturase is shown in SEQ ID NO:2. The subject matter of the invention comprise all nucleic acid sequences which, taking into consideration the degeneration of the genetic code, code for the protein sequence SEQ ID NO:2.
Preferred vectors are DNA vectors in which the DNA sequence (a) which codes for the protein phytoene desaturase, the pds gene, is preferably derived from H. pluvialis and the resistance-conferring amino acid substitution has been introduced by means of directed mutagenesis. Especially preferred vectors are those which code for the protein H. pluvialis phytoene desaturase which has an amino acid substitution from leucine to arginine at position 504 of its amino acid sequence (SEQ ID NO:3).
The amino acid substitution according to the invention from leucine to arginine at position 504 of the H. pluvialis PDS protein corresponds to the Leu437Arg substitution in Synechococcus PCC7942 (FIG. 2). For this purpose, the Leu codon “CTG” is replaced by the Arg codon “CGC” by directed mutagenesis. This mutation imparts, to the mutants, the highest known resistance factor to norflurazon. The resistance which has been conferred imparts, to the mutants according to the invention, a resistance to norflurazon which is up to 70-fold higher in comparison with the wild type. In accordance with the invention, norflurazon concentrations of 0.5-50 μm are used as selection substance.
Further positions in the amino acid sequence of H. pluvialis which are preferred for an amino acid substitution are arginine 264, leucine 388, valin 465 (FIG. 3). In Synechococcus, a resistance-mediating effect is known for the amino acid substitution at homologous positions.
Preferred are vectors which mediate, to transformants, a resistance to herbicides, in particular bleaching herbicides, and especially preferably norflurazon.
Within the scope of the invention, multiple cloning site (MCS), also referred to as polylinker, is understood as meaning a region in a nucleic acid sequence which has a large number of different utilizable cleavage sites for restriction endonucleases without the function of other elements of the nucleic acid sequence being adversely affected when a restriction hydrolysis takes place. Preferably, the DNA molecule is hydrolyzed only at one position by restriction endonucleases, whose cleavage sites are defined in the MCS. Examples of conventional MCSs are well known to the skilled worker from commercially available vectors and plasmids. Within the meaning of the present invention, MCS is furthermore understood as meaning any restriction cleavage site within a vector which can be used in order to clone in, into the vector sequence, any DNA sequences in a directed or undirected fashion without in the process adversely affecting other functional elements according to the invention of the vector.
An embodiment, of an MCS, which is preferred in accordance with the invention is shown in SEQ ID NO:4. In a preferred embodiment, the DNA vector according to the invention has the sequence SEQ ID NO:5 (see also FIG. 4).
The present invention preferably relates to those DNA vectors which comprise, as any DNA sequence to be cloned in, a coding sequence in the multiple cloning site.
Coding sequence is understood as meaning any DNA sequence which codes for a complete active protein or a protein fragment which has biological activity.
Especially preferred are coding sequences of plant origin. Especially preferred are furthermore coding sequences which comprise at least one promoter sequence. In this context, preferred promoters are those which make possible a constitutive transcription or expression of the coding sequences which are under their control. A preferred promoter is the β-tubulin promoter. Particularly preferred are the promoter sequences which are selected from the group consisting of H. pluvialis promoters of the actin gene (SEQ ID NO:6) and of the Rubisco gene (SEQ ID NO:7).
The invention furthermore relates to DNA vectors in which the coding sequence comprises a functional gene to be expressed, in addition to at least one promoter gene. Especially preferred in this context are DNA vectors which comprise a coding sequence which is selected from the group consisting of carotenoid biosynthesis genes, astaxanthin biosynthesis genes and isoprenoid biosynthesis genes. Particularly preferred are gene sequences of β-carotene ketolase, carotenoid hydroxylase, ζ-carotene desaturase, phytoene synthase, leucopene cyclase, deoxyxylulose synthase and 1-deoxy-xylose 5-phosphate reductoisomerase (Berthold et al., 2002, Hallmann and Sumper, 1996, Mahmoud and Croteau, 2001).
The invention furthermore relates -to the use of the vector according to the invention for transforming eukaryotic cells, in particular single-celled plant cells. It is especially preferred to use the vector according to the invention for the transformation of algal cells, especially. H. pluvialis cells. Within the scope of the invention, transformation is understood as meaning the introduction of foreign DNA into an organism.
The use of the vector according to the invention as selective marker for transformation purposes is also a subject matter of the invention. Especially preferred in this context is the use as dominant selective marker.
A selective marker within the meaning of the present invention mediates, to a transformed organism, a property by means of which this organism can be distinguished readily from untransformed organisms of the same species. A dominant-selective marker is understood as meaning a marker which mediates a property by means of which selection pressure can be exerted within the species so that, in a population of transformed and untransformed organisms of the same species, only the transformed organisms are viable. By adding norflurazon to growth media it is possible, for example, to select H. pluvialis cells which have been successfully transformed with the vector according to the invention of SEQ ID NO:4 from untransformed cells which are not viable under these conditions. The selection of the transformants is accomplished in accordance with the invention with norflurazon concentrations of 0.5-50 μm. The selection can be accomplished in liquid culture or by adding the herbicide to nutrient media plates.
Using mutated PDS as the first dominant selective marker for the transformation of eukaryotes, in particular algae such as H. pluvialis, the present invention provides an important contribution to the biotechnological utilization of eukaryotes, in particular algae such as H. pluviadis. For example, the vector according to the invention can be used with or without insertion of a coding sequence into the MCS in order to influence, or to modify, carotenoid biosynthesis in transformants.
The invention furthermore relates to a method of transforming eukaryotic cells using a vector according to the invention. Such transformation methods are known to the skilled worker. They comprise, for example, the use of PEG, glass beads, electroporation and micro-particle bombardment. Especially preferred in accordance with the invention is a method in which the transformation is carried out by means of particle bombardment. A preferred embodiment in this context is the particle bombardment with tungsten or gold particles 0.4 to 1.7 μm in size which have previously been coated with vector DNA according to the invention, carried out at a pressure of from 500 to 2500 psi and in vacuo. After the transformation, the cells are preferably regenerated in OHA liquid medium (2.42 g Tris-acetate pH 6.8) with shaking overnight in the dark. The cells are then plated on OHA plates under selection pressure with 0.7% strength OHA agarose. Transformants can be observed after 3-4 weeks under light-dark-intervals of equally long intervals of light (15-25 μE*m−2*s−1) and dark (in each case 6-12 h). The transformation efficacy is approximately 1*10−4 to 10*10−8 cells/μg DNA, preferably 1*10−6 cells/μg DNA.
The invention additionally comprises a transgenic plant cell and its progeny which, after transformation with the DNA vector according to the invention, is characterized in that it features an incorporation of the introduced DNA into the nuclear genome. Preferred in this context are transgenic plant cells and their progeny which display a single or multiple incorporation of the introduced DNA into the nuclear genome. Particularly preferred are transgenic plant cells and their progeny in which the introduced gene is expressed constitutively.    SEQ ID NO:1: Nucleic acid sequence of the pds gene which codes for the protein H. pluvialis phytoene desaturase    SEQ ID NO:2: Protein sequence of the H. pluvialis phytoene desaturase    SEQ ID NO:3: Protein sequence of the phytoene desaturase with Leu504 Arg amino acid substitution    SEQ ID NO:4: Nucleic acid sequence of the MCS of the vector Plat-pdsMod4.1 according to the invention    SEQ ID NO:5: Nucleic acid sequence of the preferred DNA vector Plat-pdsMOD4.1    SEQ ID NO:6: Nucleic acid sequence comprising the actin promoter (SmaI fragment). The nucleic acid sequence comprises coding regions with introns, exons and the promoter sequence, which is indicated (−).    SEQ ID NO:7: Nucleic acid sequence comprising the rbsc promoter (Rubisco small subunit) (Pstl fragment). The nucleic acid sequence comprises coding regions with introns, exons and the promoter sequence, which is indicated.